This invention relates generally to a novel myb-like protein that interacts with cyclin D. The interaction involves the regulation of RNA transcription. The invention relates to the protein, polypeptide, including biologically active or antigenic fragments thereof, and analogs and derivatives thereof, and to methods of making and using the same, including diagnostic and therapeutic uses. The invention further includes the corresponding amino acid and nucleotide sequences.
The cell cycle for growing cells can be divided into two periods: (1) the cell division period, when the cell divides and separates, with each daughter cell receiving identical copies of the DNA; and (2) the period of growth, known as the interphase period. For the cell cycle of eucaryotes, the cell division period is labeled the M (mitotic) period. The interphase period in eucaryotes is further divided into three successive phases: G1 (gap 1) phase, which directly follows the M period; S (synthetic) phase, which follows G1; and G2 (gap 2) phase, which follows the S phase, and immediately precedes the M period. During the two gap phases no net change in DNA occurs, though damaged DNA may be repaired. On the other hand, throughout the interphase period there is continued cellular growth and continued synthesis of other cellular components. Towards the end of the G1 phase, the cell passes a restrictive (R) point and becomes committed to duplicate its DNA. At this point, the cell is also committed to divide. During the S phase, the cell replicates DNA. The net result is that during the G2 phase, the cell contains two copies of all of the DNA present in the G1 phase. During the subsequent M period, the cells divide with each daughter cell receiving identical copies of the DNA. Each daughter cell starts the next round of the growth cycle by entering the G1 phase.
The G1 phase represents the interval in which cells respond maximally to extracellular signals, including mitogens, anti-proliferative factors, matrix adhesive substances, and intercellular contacts. Passage through the R point late in G1 phase defines the time at which cells lose their dependency on mitogenic growth factors for their subsequent passage through the cycle and, conversely, become insensitive to anti-proliferative signals induced by compounds such as transforming growth factor, cyclic AMP analogs, and rapamycin. Once past the R point, cells become committed to duplicating their DNA and undergoing mitosis, as noted above, and the programs governing these processes are largely cell autonomous.
In mammalian cells, a molecular event that temporally coincides with passage through the R point is the phosphorylation of the retinoblastoma protein (RB). In its hypophosphorylated state, RB prevents the cell from exiting the G1 phase by combining with transcription factors such as E2F to actively repress transcription from promoters containing E2F binding sites. However, hyperphosphorylation of RB late in G1 phase prevents its interaction with E2F, thus allowing E2F to activate transcription of the same target genes. As many E2F-regulated genes encode proteins that are essential for DNA synthesis, RB phosphorylation at the R point helps convert cells to a pre-replicative state that anticipates the actual G1/S transition by several hours. Cells that completely lack the RB function have a reduced dependency on mitogens but remain growth factor-dependent, indicating that cancellation of the RB function is not sufficient for passage through the R point.
Phosphorylation of RB at the R point is initially triggered by holoenzymes composed of regulatory D-type cyclin subunits and their associated cyclin-dependent kinases, CDK4 and CDK6. The D-type cyclins are induced and assembled into holoenzymes as cells enter the cycle in response to mitogenic stimulation. Acting as growth factor sensors, they are continuously synthesized as long as mitogenic stimulation continues, and are rapidly degraded after mitogens are withdrawn. In fibroblasts, inhibition of cyclin D-dependent CDK activity prior to the R point, either by microinjection or by scrape loading of antibodies directed against cyclin D1 or by expression of CDK4 and CDK6 inhibitors (INK4 proteins) prevents entry into S phase. However, such manipulations have no effect in cells lacking functional RB, implying that RB is the only substrate of the cyclin D-dependent kinases whose phosphorylation is necessary for exiting, the G1 phase.
Since RB-mediated controls are not essential to the cell cycle per se it is difficult to understand why mammalian cells contain three distinct D-type cyclins (D1, D2, and D3), at least two cyclin D-dependent kinases (CDK4 and CDK6), and four INK4 proteins, all, purportedly, for the sole purpose of regulating RB phosphorylation. This apparent redundancy has been explained as a method to govern transitions through the R point in different cell types responding to a plethora of distinct extracellular signals.
Alternatively, cyclin D-dependent kinases, or the cyclins alone could also be involved in the regulation of RB-independent events, perhaps linking them temporally to cell cycle controls. One mechanism for this regulation could involve the direct interaction between a cyclin, such as a D-type cyclin, and a specific transcription factor, which would allow the cyclins to regulate gene expression in an RB-independent manner. However, up until now, no such RB-independent transcription factor has been identified.
The citation of any reference herein should not be deemed as an admission that such reference is available as prior art to the instant invention.
The present invention provides a new, cyclin D-associated transcription factor. The transcription factor is an amino acid polymer which specifically binds D-type cyclins in vitro, specifically binds a DNA nucleotide sequence, and is involved in the regulation of genes that prevent cell proliferation. In one embodiment the cyclin D-associated transcription factor is a substrate of cyclin D2-CDK4 kinase. In another embodiment, the transcription factor consists of about 760 amino acids.
More particularly, the present invention includes an amino acid polymer that has a binding affinity for one or more D-type cyclins, and one or more of the following characteristics in addition to the property described above:
(1) The relative binding affinity of the amino acid polymer for cyclin D2, as compared to that for a cyclin D2 mutant that is disrupted in an amino-terminal LEU-X-CYS-X-GLU pentapeptide (SEQ ID NO:9), is minimally less disparate than the relative binding affinity of retinoblastoma protein for cyclin D2 as compared to that for the same cyclin D2 mutant.
(2) The amino acid polymer remains able to detectably interact with a cyclin D2 mutant, containing substitutions in the amino-terminal LEU-X-CYS-X-GLU pentapeptide (SEQ ID NO:9), under conditions where the binding of retinoblastoma protein to that same cyclin D2 mutant is essentially undetectable.
(3) The amino acid polymer binds preferentially to a specific DNA nucleotide sequence.
(4) The amino acid polymer is a substrate of the cyclin D2-CDK4 complex.
(5) The amino acid polymer contains three a typical tandem myb repeats.
(6) D-type cyclins bind less avidly to the amino acid polymer than to retinoblastoma protein, both in vitro and in Sf9 cells.
(7) Cyclin D-CDK4-dependent phosphorylation of retinoblastoma protein proceeds to a much higher stoichiometry than the comparative phosphorylation of the amino acid polymer under standard conditions for cyclin D-CDK4 kinase reactions.
(8) Cyclin D-dependent kinases phosphorylate the amino acid polymer at an a typical recognition sequence.
(9) The amino acid polymer binds preferentially to nucleic acids containing the nonamer sequence CCCGTATGT.
(10) Relative to the cyclin D-CDK4 complex, cyclin E-CDK2 complexes phosphorylate the amino acid polymer poorly, if at all.
(11) A catalytically-inactive CDK4 does not enter into a stable ternary complex with cyclin D and the amino acid polymer under conditions where retinoblastoma protein, cyclin D and the identical catalytically-inactive CDK4 form stable ternary complexes.
(12) Cyclin D mutants which do not bind to CDK4 still interact with the amino acid polymer at unreduced efficiency.
(13) Overexpression of the amino acid polymer can arrest the cell cycle in G1 phase preventing proliferating cells from replicating their chromosomal DNA.
(14) The activity of the amino acid polymer in arresting cell growth in G1 phase depends both upon its ability (a) to bind DNA and (b) to activate transcription, and mutants defective in either of these properties are unable to prevent cells from entering S phase.
(15) Enforced transient expression of cyclin D2 or D2-CDK4 in mammalian cells negatively regulate the ability of the amino acid polymer to transactivate reporter gene expression.
(16) The amino acid polymer activates transcription more readily in quiescent cells lacking cyclin D expression, than in proliferating cells containing cyclin D.
(17) Enforced expression of cyclin D-CDK4 does not influence the stability of the amino acid polymer.
(18) Enforced expression of cyclin D-CDK4 does not influence the ability of the amino acid polymer to preferentially localize to the nucleus of transfected mammalian cells. Although any person having skill in the art would know that many of the above characteristics may be determined in vitro, the present invention includes the same or analogous characteristics that are determined either in situ or in vivo.
(19) Cyclin D binding to the amino acid polymer inhibits its ability to induce cell cycle arrest.
In one aspect of the present invention the amino acid polymer binds preferentially to a DNA nucleotide sequence, termed herein the cyclin D-associated transcription factor binding site or the DMP1 binding site. In a more specific embodiment, the binding site has the core trinucleotide sequence GTA. In some embodiments the nucleotide sequence contains a nonamer consensus sequence CCCG(G/T)ATGT. In other embodiments the nucleotide sequences contain multiple concatamers of the nonamer consensus sequence. In preferred embodiments the nucleotide sequence contains the nonamer consensus sequence CCCGTATGT.
The present invention provides an isolated amino acid polymer obtained from animal cells, produced recombinantly, or prepared by chemical synthesis. In preferred embodiments the amino acid polymer is mammalian. In a more preferred embodiment the amino acid polymer is a primate protein. In the most preferred embodiments, the amino acid polymer is human. In a specific example, the amino acid polymer is obtained from a murine cell and has the amino acid sequence of SEQ ID NO:1. In a related embodiment the amino acid polymer has an amino acid sequence of SEQ ID NO:1 having one or more conservative amino acid substitutions. In another embodiment, the amino acid polymer is obtained from a human cell and contains the amino acid sequence of SEQ ID NO:24. In a related embodiment, the amino acid polymer has an amino acid sequence of SEQ ID NO:24 having one or more conservative amino acid substitutions. In a preferred embodiment, the amino acid polymer has the amino acid sequence of SEQ ID NO:29. In a related embodiment, the amino acid polymer has an amino acid sequence of SEQ ID NO:29 having one or more conservative amino acid substitutions. In a related embodiment the isolated amino acid polymer is obtained from a human cell, is encoded on human chromosome 7 at a position which corresponds to 7q21, and contains about 760 amino acids including the 262 amino acids of SEQ ID NO:24.
The present invention relates to the identification and elucidation of a direct interaction between D-type cyclins and a novel myb-like transcription factor termed herein DMP1. This novel factor has been found to specifically interact with cyclin D2. This present invention also describes the regulation of gene expression by D-type cyclins, and other related methods of use, in an RB-independent manner.
As shown in the Examples, infra, DMP1 includes a central DNA-binding domain containing three atypical myb repeats flanked by highly acidic segments located at its amino- and carboxylterminal ends. The present invention includes amino acid sequences coding for DMP1, including amino acid sequences containing conservative substitutions of such amino acids.
The present invention also includes peptides containing portions of amino acid polymers of the present invention, including fragments of the amino acid polymers. One such peptide corresponds to the DNA-binding domain of the amino acid polymer of the present invention. In one specific embodiment of this type, the peptide has an amino acid sequence of SEQ ID NO:16. In another such embodiment the peptide has an amino acid sequence of SEQ ID NO:16 having one or more conservative amino acid substitutions. The present invention also includes a peptide that corresponds to the transactivation domain of the amino acid polymer of the present invention. In one specific embodiment of this typed the peptide has an amino acid sequence of SEQ ID NO:18. In another such embodiment the peptide has an amino acid sequence of SEQ ID NO:18 having one or more conservative amino acid substitutions. In yet another specific embodiment of this type, the peptide has an amino acid sequence of SEQ ID NO:20. In still another such embodiment the peptide has an amino acid sequence of SEQ ID NO:20 having one or more conservative amino acid substitutions. In yet another specific embodiment of this type, the peptide has an amino acid sequence consisting of SEQ ID NO:18 and SEQ ID NO:20. In still another such embodiment the peptide consisting of an amino acid sequence of SEQ ID NO:18 and SEQ ID NO:20, having one or more conservative amino acid substitutions. The present invention further includes a peptide that corresponds to the D-type cyclin binding domain of the amino acid polymer of the present invention. In one specific embodiment of this type, the peptide has an amino acid sequence of SEQ ID NO:22. In another such embodiment the peptide has an amino acid sequence of SEQ ID NO:22 having one or more conservative amino acid substitutions. DNA and RNA nucleotide sequences that encode for the amino acid polymers of the present invention, and methods of use thereof are also included.
One method of the invention includes the use of DMP1 as a transcription factor due to its specificity in binding to oligonucleotides containing the nonamer consensus sequence CCCG(G/T)ATGT. A recombinant expression vector comprising the foregoing consensus sequence operably associated with a gene for expression can be prepared. In this aspect of the invention, DMP1 activates the transcription of a heterologous gene including reporter genes driven by a minimal promoter containing concatamerized DMP1 binding sites. If necessary, the invention provides for expression of DMP1 with the foregoing expression vector in order to enhance DMP1-mediated transcription from the expression vector.
Another aspect of the present invention includes fusion proteins. All of the amino acids polymers and peptides of the present invention may contain a fusion peptide (e.g. FLAG) or protein (e.g. GST or green fluorescent protein). Such examples include GST-DMP1 or green fluorescent protein-DMP1. These fusion proteins may be used to bind directly to D-type cyclins in vitro, including radiolabeled D-type cyclins.
In addition, all of the nucleic acids of the present invention can be combined with heterologous nucleotide sequences. For example, the present invention provides a nucleic acid consisting of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:29 and a heterologous nucleotide sequence. Such a nucleic acid can encode a fusion peptide and fusion protein discussed above, for example.
In still another aspect of the invention, complexes between full-length DMP1 and D-type cyclins readily form in intact Sf9 insect cells engineered to co-express both proteins under baculovirus vector control.
A further aspect of the invention includes the use of detectable labels, such as but not limited to a protein including an enzyme, a radioactive element, a bioluminescent, a chromophore that absorbs in the ultraviolet and/or visible and/or infrared region of the electromagnetic spectrum; and a fluorophore. The present invention includes an amino acid polymer labeled with such a detectable label. The present invention also includes reporter genes encoding proteins that contain detectable labels, such as green fluorescent protein, or an 35S-labeled protein, can interact with a label such as a labeled antibody or can catalyze a reaction that gives rise to a detectable signal, such as the bioluminescence catalyzed by firefly luciferase. The present invention also includes antibodies to all of the amino acid polymers of the instant invention. The antibodies of the present invention may be either polyclonal or monoclonal. Either type of antibody can further comprise a detectable label described above. In one such embodiment, the antibody is raised against the amino acid polymer of SEQ ID NO:29, or antigenic fragment thereof.
Naturally, in addition to the transcription factor, the present invention provides nucleic acids that contain nuclectide sequences or degenerate variants thereof, which encode the amino acid polymers of the present invention. In this aspect of the invention the nucleotide sequence can contain the coding region of the DNA sequence of SEQ ID NO:2 or an RNA sequence corresponding to SEQ ID NO:3; or a DNA sequence encoding a full length human DMP1 containing the nucleic acid sequence SEQ ID NO:25 or an RNA sequence encoding a full length human DMP1 containing the nucleic acid sequence SEQ ID NO:26. In one embodiment, the nucleic acid encodes a full length human DMP1 having the amino acid sequence of SEQ ID NO:29. In a preferred embodiment, the nucleic acid has a DNA sequence containing the coding region of SEQ ID NO:28, or the RNA, sequence containing the coding region of SEQ ID NO:30. In a related embodiment the nucleic acid encodes an isolated amino acid polymer which is encoded on human chromosome 7 at a position which corresponds to 7q21, and contains about 760 amino acids, including the 262 amino acids of SEQ ID NO:24.
In addition, the present invention also includes a nucleic acid encoding a peptide that corresponds to the DNA-binding domain of the amino acid polymer of the present invention. In one such embodiment the nucleic acid encodes a peptide having an amino acid sequence of SEQ ID NO:16, or SEQ ID NO:16 having one or more conservative amino acid substitutions. In one specific embodiment of this type, the nucleic acid sequence is SEQ ID NO:17. The present invention also includes a nucleic acid encoding a peptide that corresponds to the transactivation domain of the amino acid polymer of the present invention. In one such embodiment the nucleic acid encodes a peptide having an amino acid sequence of SEQ ID NO:18, or SEQ ID NO:18 having one or more conservative amino acid substitutions. In one specific embodiment of this type, the nucleic acid sequence is SEQ ID NO:19. In yet another specific embodiment of this type, the nucleic acid encodes a peptide having an amino acid sequence of SEQ ID NO:20, or SEQ ID NO:20 having one or more conservative amino acid substitutions. In one specific embodiment of this type, the nucleic acid sequence is SEQ ID NO:21. In yet another specific embodiment of this type, the nucleic acid encodes a peptide having an amino acid sequence consisting of SEQ ID NO:18 and SEQ ID NO:20 or consisting of an amino acid sequence of SEQ ID NO:18 and SEQ ID NO:20 having one or more conservative amino acid substitutions. In one specific embodiment of this type, the nucleic acid sequence consists of SEQ ID NO:19 and SEQ ID NO:21. The present invention further includes a nucleic acid encoding a peptide that corresponds to the D-type cyclin binding domain of the amino acid polymer of the present invention. In one specific embodiment of this type, the nucleic acid encodes a peptide having an amino acid sequence of SEQ ID NO:22, or SEQ ID NO:22 having one or more conservative amino acid substitutions. In one specific embodiment of this type, the nucleic acid sequence is SEQ ID) NO:23.
Nucleic acids containing sequences complementary to these sequences, or nucleic acids that hybridize to any of the foregoing nucleotide sequences under standard hybridization conditions are also part of the present invention. In a preferred embodiment, the nucleic acids hybridize to the foregoing nucleotide sequences under stringent conditions.
In preferred embodiments the nucleic acid is a recombinant DNA sequence that is operatively linked to an expression control sequence.
Another aspect of the invention includes methods for detecting the presence or activity of the amino acid polymer of the invention in a biological sample that is suspected to contain the amino acid polymer. These methods include steps of contacting a biological sample with a nucleotide probe under conditions that allow binding of the nucleotide probe to the amino acid polymer to occur, and then detecting whether that binding has occurred. In a specific embodiment, the nucleotide probe contains the sequence CCCGTATGT. The detection of the binding indicates the presence or activity of the amino acid polymer in the biological sample. The nucleotide probe may be labeled with a detectable label as described above. In a preferred embodiment of this aspect of the invention the nucleotide probe has a detectable label containing the radioactive element, 32P, and the detecting step includes performance of an electrophoretic mobility shift assay. In another specific embodiment, the DMP1 binding site may be used to isolate a DMP1 amino acid polymer by specific affinity binding. More particularly, the CCCGTATGT nonanucleotide may be used to) isolate a mammalian DMP1 polypeptide.
Another aspect of the present invention includes methods of activating selective transcription of a heterologous gene operably associated with a DNA sequence to which the present transcription factor binds in mammalian cells. These methods include the step of recombinantly fusing a control unit comprising the nucleotide sequence, e.g., CCCGTATGT, to a selected gene forming a controllable transcript, and transfecting a mammalian cell with the recombinant gene. In some embodiments of the invention, the endogenous transcription factor of the invention in the mammalian cell will be sufficient to activate selective transcription of the heterologous gene. In other embodiments the basal level of the amino acid polymer in the mammalian cells used will be insufficient to activate detectable transcription of the recombinant heterologous gene. In these other embodiments, the amino acid polymer of the present invention may be added to the mammalian cell, e.g., by microinjection or transfection, with an expression vector comprising the transcription factor gene into the cells, thereby activating transcription of the selected gene.
The present invention also includes the use of an oligonucleotide comprising the DMP1 binding site, e.g., the nonamer sequence CCCGTATGT, as a competitive inhibiter for blocking the activation of selective transcription by the amino acid polymer.
The present invention also includes an antisense nucleic acid against an mRNA coding for the amino acid polymer of the present invention and is therefore capable of hybridizing to the mRNA. The antisense nucleic acid may be either an RNA or a DNA, preferably containing a phosphodiester analog.
In a further aspect, the present invention provides a transgenic animal comprising the expression vector which provides for increased or xe2x80x9csuper-xe2x80x9d expression of the cyclin D-associated transcription factor homologously recombined in a chromosome or a cyclin D-associated transcription factor that no longer binds a cyclin D, such as cyclin D 1. In a related embodiment, the present invention provides a transgenic animal in which the gene encoding an amino acid polymer of the present invention, such as murine DMP1, has been disrupted so as to be unable to express a functional transcription factor. Disruption of expression can be achieved by (i) knocking out the gene; (ii) introducing a null or non-sense mutation in the gene; (iii) deleting the regulatory sequences necessary for effective transcription of the gene; and (iv) introducing a mutation into the gene that results in expression of an inactive protein, e.g., a protein which fails to bind to DNA, to the DMP1 binding site on DNA, to transactivate genes under the control of a DMP1-responsive promoter, or any combination of the foregoing.
The present invention also includes methods of identifying genes that are under the control of DMP1-responsive promoters. Such genes play an important role in cell regulation, and more particularly in hindering the proliferation of the cell.
The present invention also includes drug assays for identifying drugs that antagonize or agonize the effect of DMP1 on genes under the control of a DMP1-responsive promoter. One such method is for identifying a drug that inhibits the transactivation of a gene by DMP1 in situ, comprising cotransfecting a cell with a first expression vector containing a reporter gene under the control of a promoter responsive to DMP1, and a second expression vector encoding DMP1, or a fragment thereof capable of transactivating the promoter. A potential drug is then contacted with the cell, and the expression of the reporter gene is detected. A drug is identified when the expression of the reporter gene is decreased. In preferred embodiments of this type, the identified drug prevents the detectable expression of the reporter gene
In one particular embodiment of this type, the second expression vector encodes an amino acid polymer having the amino acid sequence of SEQ ID NO:1, or SEQ ID NO:1 having one or more conservative amino acid substitutions. In another embodiment, the second expression vector encodes an amino acid polymer having the amino acid sequence of SEQ ID NO:29, or SEQ ID NO:29 having one or more conservative amino acid substitutions. In yet another embodiment of this type the second expression vector encodes a fragment of DMP1 having an amino acid sequence of SEQ ID NO:18, or SEQ ID NO:18 having one or more conservative amino acid substitutions. In still another embodiment, the promoter is an artificial DMP1-responsive promoter. In a preferred embodiment of this type, the artificial promoter consists of 8xc3x97BS2 (CCCGTATGT) inserted into the XhoI-SmaI sites of pGL2 (Promega) 5xe2x80x2 to a minimal simian virus 40 (SV40) early promoter driving the reporter gene. In another preferred embodiment, the reporter gene is firefly luciferase. In one embodiment, the cell is a mammalian cell, such as a mouse NIH-3T3 fibroblast. In preferred embodiments, the mammalian cell is a human cell. The potential drug may be selected by rational design, such as an analog of a cyclin, or an analog to the DNA-binding domain of DMP1, as described herein. Alternatively, the potential drug can be randomly obtained from a drug library, including from one described herein.
The present invention also includes in vitro assays to identify drugs that will bind to the cyclin binding domain of DMP1. In a preferred embodiment the cyclin binding domain has an amino acid sequence of SEQ ID NO:22, or SEQ ID NO:22 having one or more conservative amino acid substitutions. Such drugs can either inhibit DMP1 by acting as an analog of the cyclins; or alternatively, the drug can prevent the inhibition of the cyclin-dependent inhibition of DMP1 by preventing a cyclin from binding to DMP1 while not interfering with the transactivation properties of DMP1.
In one such embodiment, the method comprises placing the cyclin binding domain of DMP1 on a solid support, contacting the cyclin binding domain of DMP1 with a potential drug that is labeled, washing the solid support, and detecting the potential drug associated with the cyclin binding domain of DMP1. A potential drug is identified as a drug if it is detected with the cyclin binding domain of DMP1. The method can further comprise a step of washing the solid support with an excess of a cyclin, such as cyclin D2, prior to the detection step. In this case a potential drug is identified as a drug, if washing with cyclin hinders or prevents the detection of the labeled drug with cyclin binding domain of DMP1. Again the potential drug may be selected by rational design, such as an analog of a cyclin, or alternatively the potential drug can be randomly obtained from a drug library, including from one described herein.
An identified drug cm then be assayed in situ, as described above to determine whether it enhances or diminishes the transactivation of a reporter gene under the control of a DMP1-responsive promoter. A drug is selected as an antagonist of DMP1 when the expression of the reporter gene is decreased. A drug is selected as an agonist of DMP1 when the expression of the reporter gene is increased. The method can further comprise coexpressing a cyclin, such as cyclin D2, and DMP1 in a cell and determining whether the drug prevents the inhibitory effect of the cyclin. Such a drug is selected as an agonist of DMP1, if it can hinder and/or prevent the inhibitory effect of the cyclin.
An additional embodiment includes a method of determining the effect of the drug on a CDK comprising contacting the identified drug with a CDK and performing a cyclin-CDK linase assay on an appropriate substrate, such as retinoblastoma protein (as described herein) in the absence of a cyclin, wherein a drug is selected if the kinase assay is negative. The cyclin-CDK kinase assay is next performed with cyclin, the CDK, appropriate substrate and an excess of the drug. A drug is selected which does not interfere with the phosphorylation of the appropriate substrate by the cyclin-CDK.
Another aspect of the present invention includes a method of inducing cell cycle arrest in a eukaryotic cell without provoking cell death comprised of introducing DMP1 or an active DMP1 fragment into the cell. In this case an active DMP1 fragment acts by inducing the transcription of ARF-p19. In a particular embodiment of this type, introducing DMP1 or an active DMP1 fragment into the cell is performed by placing the DMP1 polypeptide or an active DMP1 fragment into the cell. In another embodiment, introducing DMP1 or an active DMP1 fragment into the cell is performed by placing an expression vector comprising a nucleic acid encoding the DMP1 polypeptide or an active DMP1 fragment into the cell.
The present invention further provides isolated nucleic acids comprising ARF-p19 promoters and fragments thereof. In one particular embodiment, the fragment comprises the nonamer sequence CCCGGATGC (SEQ ID NO:33). In another embodiment the ARF-p19 promoter comprises SEQ ID NO:34. In a related embodiment the ARF-p19 promoter comprises nucleotides xe2x88x92225 to +56 of SEQ ID NO:34. In still another embodiment the fragment comprises the nonamer sequence GACGGATGT (SEQ ID NO:35). The present invention also provides expression vectors having a transcription control sequence comprising the ARF-p19 promoters and fragments thereof operably associated with a recombinant gene or a cassette insertion site for a recombinant gene.
Yet another aspect the present invention provides methods of preventing abnormal cell growth in a eukaryotic cell. In a particular embodiment of this type, the method comprises administering an effective amount of DMP1 or an active DMP1 fragment to the cell. In this case an active DMP1 fragment acts by inducing the transcription of ARF-p19. In another embodiment, the administration of an effective amount of DMP1 or the active DMP1 fragment comprises administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and DMP1 or the active DMP1 fragment. In still another embodiment, the method of administering an effective amount of DMP1 or the active DMP1 fragment comprises administering an expression vector comprising a nucleic acid encoding DMP1 or the active DMP1 fragment.
The present invention also provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and DMP1 or an active DMP1 fragment. As described above, the active DMP1 fragment can act by inducing the transcription of ARF-p19.
The present invention further provides methods for diagnosing a biological sample. In one such embodiment, the biological sample comprises a eukaryotic cell suspected of being cancerous due to a mutation, deletion, or insertion in an endogenous nucleic acid encoding DMP1. A particular embodiment of this type comprises preparing a DNA or RNA sampler from the cell and detecting the mutation, the deletion, or the insertion with the nucleotide sequence of SEQ ID NO:28: or a portion thereof. When the mutation, the deletion, or the insertion is detected, the presence of the mutation, the deletion, or the insertion of the endogenous nucleic acid encoding DMP1 is diagnosed. In one such embodiment of this type, the portion of SEQ ID NO:28 is a nucleotide probe. In another embodiment, the portion of SEQ ID NO:28 is a primer.
In a related embodiment, the biological sample being diagnosed comprises a eukaryotic cell suspected of being cancerous due to a significant decrease in its ability to express wild type DMP1. A particular embodiment of this type comprises preparing a sample from the cell and detecting wild type DMP1 by cross reacting the sample with an antibody for wild type DMP1. When the amount of cross-reaction with the antibody for wild type DMP1 is significantly lower than that found for a corresponding wild type cell, the cell is diagnosed as having a significant decrease in its ability to express wild type DMP1.
The present invent further provides methods for identifying an agent that modulates the ability of DMP1 to transactivate an ARF-p19 promoter. One such method comprises contacting an agent with a cell which contains DMP1 and a marker gene under the transcriptional control of an ARF-promoter that can bind DMP1. The amount of expression of the marker gene is determined. The agent is then contacted with a cell in the absence of DMP1 and the amount of expression of the marker gene is again determined. An agent is identified as modulating the ability of DMP1 to transactivate the ARF-p19 promoter when the amount of marker gene expressed is different in the presence of the agent than in its absence, and wherein in the absence of DMP1 the marker gene is not expressed or is expressed at a basal level. In a particular embodiment, the agent has a molecular weight of less than 3 kilodaltons. In another embodiment, the agent is not a naturally occurring compound.
In one embodiment the ARF-p19 promoter that binds DMP1 comprises the nucleotide sequence of SEQ ID NO:33. In another embodiment the ARF-p19 promoter that binds DMP1 comprises the nucleotide sequence of nucleotides xe2x88x92225 to +56 of SEQ ID NO:34. In yet another embodiment the ARF-p19 promoter that binds DMP1 comprises the nucleotide sequence of SEQ ID NO:34. In still another embodiment the ARF-p19 promoter that binds DMP1 comprises the nucleotide sequence of SEQ ID NO:35. In yet another embodiment the ARF-p19 promoter that binds DMP1 comprises the nucleotide sequence of SEQ ID NO:36 or a fragment thereof that binds DMP1.
The present invention also provides a method for identifying an agent that can mimic the ability of DMP1 to transactivate an ARF-p19 promoter. One such embodiment comprises contacting an agent with a cell that does not contain active DMP1 (i.e., active DMP1 is a form of DMP1 that binds the ARF-p19 promoter) but does contain a marker gene under the transcriptional control of an ARF-p19 promoter that binds DMP1. The amount of marker gene expressed is determined and an agent is selected when the amount of marker gene expressed is increased in the presence of the agent. The selected agent is then contacted with a cell containing an ARF-p19 promoter that does not bind DMP1 and the amount of marker gene expressed is determined. An agent is selected when the amount of marker gene expressed in the cell containing a marker gene under the transcriptional control of an ARF-p19 promoter that binds DMP1 is greater than the amount of marker gene expressed in the cell containing a marker gene under the transcriptional control of an ARF-p19 promoter that does not bind DMP1. In a particular embodiment, the increase in expression of the marker gene in the presence of the agent is at least 10% of that observed in the presence of DMP1. In a preferred embodiment, the increase in expression of the marker gene in the presence of the agent is at least 50% of that observed in the presence of DMP1. In a particular embodiment, the percent activity of the agent relative to DMP1 is based on gram to gram molecular weight basis. In an alternative embodiment, the percent activity of the agent relative to DMP1 is based on mole to mole basis. In a particular embodiment, the agent has a molecular weight of less than 3 kilodaltons. In another embodiment the agent is not a naturally occurring compound.
In one embodiment the ARF-p19 promoter that binds DMP1 comprises the nucleotide sequence of SEQ ID NO:33. In another embodiment the ARF-p19 promoter that binds DMP1 comprises the nucleotide sequence of nucleotides xe2x88x92225 to +56 of SEQ ID NO:34. In yet another embodiment the ARF-p19 promoter that binds DMP1 comprises the nucleotide sequence of SEQ ID NO:34. II still another embodiment the ARF-p19 promoter that binds DMP1 comprises the nucleotide sequence of SEQ ID NO:35. In yet another embodiment the ARF-p19 promoter that binds DMP1 comprises the nucleotide sequence of SEQ ID NO:36 or a fragment thereof that binds DMP1.
These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.